Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-t was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. #link# Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.After parturition, dairy cows mobilize AA from skeletal muscle to meet metabolizable protein (MP) requirements. High mobilization may compromise cow health and longer-term milk production. Postpartum diets with higher MP concentrations, improved AA profiles, or MP increased at the expense of forages rather than nonforage fiber sources may attenuate muscle catabolism; however, the molecular mechanisms responsible need investigation. We evaluated mRNA expression in the longissimus dorsi of cows fed postpartum diets differing in MP concentration, AA profile, and fiber source. From 0 to 25 d after parturition, 40 multiparous cows received the following diets (1) 13% deficient in MP (D-MP), (2) adequate in MP using primarily soy protein (A-MP), (3) adequate in MP using blends of proteins and individual AA to improve the AA profile (Blend), or (4) similar to Blend except additional protein replaced forage (Blend-fNDF). Biopsies were taken approximately -5, 7, and 25 d relative to parturition. Greater dietary MP conduce cell growth and cause autophagy.Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, Akti-1/2 (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.There has been a growing interest in cofermentation of starter cultures with probiotics in milk. In this study, we analyzed the effects of adding the single probiotics Lactobacillus casei Zhang (Zhang) or Bifidobacterium animalis ssp. lactis Probio-M8 (M8) or a combination of Zhang and M8 to starter cultures on volatile and nonvolatile metabolomic profiles after 14 d of storage at 4°C and compared using a liquid chromatography-tandem mass spectrometry (LC-MS) and GC-MS-based metabolomics approach. Principal component analysis, heatmap plots, and Spearman correlation results showed that Zhang alone had a greater effect on volatile and nonvolatile metabolomic profiles than M8 alone. The combination of Zhang and M8 had additive effects on the production of metabolites. For volatile metabolites, the levels of acetaldehyde, diacetyl, acetoin, and acetic acid were higher for the combination of Zhang and M8 compared with either single probiotic culture. Significantly increased nonvolatile components induced by adding Zhang were identified were enriched in the galactose, amino- and nucleotide sugar, fructose and mannose, purine, phenylalanine metabolism, and arginine biosynthesis pathways. The metabolism and biosynthesis of starch, sucrose, tyrosine, galactose metabolism, and aminoacyl-tRNA biosynthesis were significantly upregulated by adding the combination of Zhang and M8. This work provides a detailed insight into different effects of Zhang and M8 used alone or in combination on the volatile and nonvolatile metabolomic profiles of yogurts.Our first objective was to redesign a modified 14-sample milk calibration sample set to obtain a well-distributed range of milk urea nitrogen (MUN) concentrations while maintaining orthogonality with variation in fat, protein, and lactose concentration. Our second objective was to determine the within- and between-laboratory variation in the enzymatic spectrophotometric method on the modified milk calibration samples and degree of uncertainty in MUN reference values, and then use the modified milk calibration samples to evaluate and improve the performance of mid-infrared partial least squares (PLS) models for prediction of MUN concentration in milk. Changes in the modified milk calibration sample formulation and manufacturing procedure were made to achieve the desired range of MUN concentrations. A spectrophotometric enzymatic reference method was used to determine MUN reference values, and the modified milk calibration samples were used to calibrate 3 mid-infrared milk analyzers. The within- and between-laboratory variation in the reference values for MUN were 0.